Enzymes bring reactants to the transition state with up to 20 orders of magnitude greater efficiency than uncatalyzed reactions. Transition state structure imposed by enzymes can be determined by a combination of intrinsic kinetic isotope effects and quantum chemistry. Chemically stable analogues designed from the electrostatic maps of the experimentally determined enzymatic transition states are among the most powerful inhibitors known. Logically designed transition state analogues are in human clinical trials. Yet, the application of transition state structure to the inhibitor design and barrier crosing is in its infancy, with application to only a few reaction classes. This project will expand the utlity of transition state analysis and inhibitor design to new reaction classes. The fundamental nature of enzymatic transition states will be investigated to determine the contribution of short time scale protein vibrational motions to enzymatic transition state barrier crossing. The first aim is o target an esterase responsible for degrading a product of the sirtuin pathways, 2'-O-acetyl-ADP-ribose. This unstable ester has the chemical characteristics of a down-stream signal-transducing molecule. The esterase transition state structure will be used to design transition state analogues as inhibitors of the A2'-O-acetyl-ADP-ribose esterase. Transition state analogue design also has potential to yield a new generation of powerful inhibitors fr validated biomedical targets. Sulfonamides are p- aminobenzoate antagonists of dihydropteroate synthetase, a target for pathogenic bacteria and protozoan pathogens. The transition state structure of this pyrophosphate displacement reaction will provide new transition state information to permit the design of inhibitors for antibiotic combination therapy. The third aim relates to the enzymatic dynamic motion inexorably linked to catalysis. Experimental approaches to resolve fast (fs-ps) protein motions are needed to understand dynamic contributions to transition state barrier-crossing. On-enzyme chemistry of enzymes with increased mass provides a new analytical tool to evaluate the contribution of fast motions to barrier- crossing. The heavy enzyme approach will be applied to human purine nucleoside phosphorylase. PUBLIC HEALTH RELEVANCE: The discovery of new drugs has slowed despite exhaustive efforts in the pharmaceutical industry, a concern of the NIH and world health organizations. Enzymatic transition state analysis provides another path to powerful drugs by providing insights into the fundamental catalytic nature of enzymes as drug targets. Although transition state drug design based on experimental transition states is in its infancy, several candidates have reached clinical trials, thus, providing proof of concept for a new method of drug discovery.